title: Dissecting the effects of periplasmic chaperones on the in vitro folding of the outer membrane protein PagP.
abstract: Although many periplasmic folding factors have been identified, the mechanisms by which they interact with unfolded outer membrane proteins (OMPs) to promote correct folding and membrane insertion remain poorly understood. Here, we have investigated the effect of two chaperones, Skp and SurA, on the folding kinetics of the OMP, PagP. Folding kinetics of PagP into both zwitterionic diC12:0PC (1,2-dilauroyl-sn-glycero-3-phosphocholine) liposomes and negatively charged 80:20 diC12:0PC:diC12:0PG [1,2-dilauroyl-sn-glycero-3-phospho-(1'-rac-glycerol)] liposomes were investigated using a combination of spectroscopic and SDS-PAGE assays. The results indicate that Skp modulates the observed rate of PagP folding in a manner that is dependent on the composition of the membrane and the ionic strength of the buffer used. These data suggest that electrostatic interactions play an important role in Skp-assisted substrate delivery to the membrane. In contrast, SurA showed no effect on the observed folding rates of PagP, consistent with the view that these chaperones act by distinct mechanisms in partially redundant parallel chaperone pathways that facilitate OMP assembly. In addition to delivery of the substrate protein to the membrane, the ability of Skp to prevent OMP aggregation was investigated. The results show that folding and membrane insertion of PagP can be restored, in part, by Skp in conditions that strongly favour PagP aggregation. These results illustrate the utility of in vitro systems for dissecting the complex folding environment encountered by OMPs in the periplasm and demonstrate the key role of Skp in holding aggregation-prone OMPs prior to their direct or indirect delivery to the membrane.
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Lindsay M
authors.initials: LM
authors.lastname: McMorran
authors.forename: Alice I
authors.initials: AI
authors.lastname: Bartlett
authors.forename: Gerard H M
authors.initials: GH
authors.lastname: Huysmans
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
authors.forename: David J
authors.initials: DJ
authors.lastname: Brockwell
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008081:Liposomes; D008565:Membrane Proteins; D018832:Molecular Chaperones; C072721:Skp protein, E coli; D000217:Acyltransferases; C468520:PagP protein, E coli; C490900:SurA protein, E coli; D019696:Peptidylprolyl Isomerase
keywords: 1,2-dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol); 1,2-dilauroyl-sn-glycero-3-phosphocholine; BAM; HT PagP; His-tagged PagP; LPR; LPS; LUV; OM; OMP; PagP; Skp; SurA; diC(12:0)PC; diC(12:0)PG; folding kinetics; large unilamellar vesicle; lipid-to-protein ratio; lipopolysaccharide; membrane protein folding; outer membrane; outer membrane protein; β-barrel assembly machine